The formation of sulfenic acid groups in proteins has been recently recognized as a key event in many important biological processes. Sulfenic acids (Cys-SOH) arise from the oxidation of the thiol group of cysteine. Recent work has indicated that cellular oxidants (reactive oxygen species, ROS) are involved in cellular signaling and regulation. (See, Leonard et al., ACS Chem Biol, 2009 4(9):783-799 and references 1-3 therein).
A common reversible redox process of a protein cysteine is illustrated below. In one direction, oxidation of cysteine in the presence of ROS, such as hydrogen peroxide (H2O2), generates the corresponding sulfenic acid.

In some cases, such as with protein-tyrosine phosphatase 1B (PTP1B; EC 3.1.3.48), this oxidative modification converts the active enzyme (with a catalytic cysteine) to an inactive form containing a sulfenic acid, Cys-SOH (see, Meng et al., Mol. Cell, 2002, 9:387-399; Salmeen et al., Nature, 2003 423:769-773; and van Montfort et al., Nature, 2003 423:773-777 and references therein). Sulfenic acid formation in a protein contemplated by this invention leads to the biological activity that is inhibited by a contemplated compound as is discussed hereinafter.
A sulfinic acid generated by a reactive oxygen species such as hydrogen peroxide is often the intermediate to further modifications such as disulfide bond formation, S-glutathiolation, S-nitrosation, and sulfenyl amide formation [Paulsen et al., Chem. Rev. 2013 113:4633-4679; Jacob et al., Chem. Res. Toxicol. 2012 25:588-604; and Winterbourn et al., Free Radical Bio. Med. 2008 45:549-561]. The reversible nature of cysteine oxidation is analogous to phosphorylation, making them both well suited for regulatory posttransloational modifications. This also means that biological sulfenic acids can be a new class of potential pharmaceutical targets, especially given the growing implications of cysteine-derived sulfenic acids' role in health issues such as cancer, [Mahajan et al., J. Biol. Chem. 2013 288:11611-11620; Anastasiou et al., Science 2011 334:1278-1283; Seo et al., Proc. Natl. Acad. Sci. USA 2009 106:16163-16168] heart disease [Svoboda et al., Circ. Res. 2012 111:842-853], and scurvy [Zito et al., Mol. Cell. 2012 48:39-51].
The articles cited below illustrate and highlight the diverse scope and prevalence of sulfenic acid groups in biologically active protein systems. Li et al., J Mol Biol 2005 346:1035-1046; Hill et al., J Mol Cell Cardiol 2012 52:559-567; Brigelius-Flohe' et al., Anitoxid Redox Signal 2011 15(8):2335-2381; Seo et al., Proc Natl Adad Sci USA 2009 106(38):16163-16168; Paulsen et al., Nature Chem Biol 2012 8:57-64; Maccari et al., J Med Chem 2012 55:2-22; Roos et al., Free Rad Bio Med 2011 51:314-326; Jacob et al., Chem Res Toxicol 2012 25:588-604; Paulsen et al., ACS Chem Biol 2010 5(1):47-62; Wood et al., Trends Biochem Sci 2003 28(1):32-40; and Tonks, Nat Rev Mol Cell Biol 2006 7:833-846.
Among the known biologically-active proteins that contain/form sulfenic acids, most fall within three major classes: 1) phosphatases that are vital to the signaling pathway and the cell cycle; 2) peroxidases that play critical cellular antioxidant roles; and 3) the redox-modulated gene transcription factors that regulate protein expression level under different oxidative conditions. For example, Li et al., J. Mol. Biol. 2005 346:1035-1046 showed that M. tuberculosis encodes a 153-residue protein AhpE, which is a peroxidase of the 1-Cys peroxiredoxin (Prx) family, EC 1.11.1.15. The ahpC gene is widespread in microorganisms, and its gene product AhpC belongs to the nonheme peroxiredoxin (Prx) family, whose members are found in a wide range of biological systems, from bacteria to mammalian cells.
The importance of the Prxs, throughout living systems, is underlined by their extremely high abundance in both prokaryotic and mammalian cells. They are ubiquitous antioxidant enzymes that are expressed at high levels in cells. They are responsible for reducing a broad range of toxic peroxides and peroxinitrites (Tripathi et al., Protoplasma, 2009 235:3-15). All Prxs share a common reactive Cys residue in their N-terminal region, which is oxidized by peroxides to a cysteine sulfenic acid (Cys-SOH). The regeneration of the reduced form (back to Cys-SH) is necessary for the enzyme to regain its catalytic function. Depending on the presence or absence of other conserved cysteine residues in the sequence, Prx proteins can then be divided into 2-Cys and 1-Cys types. The 1-Cys Prx proteins are less well characterized with three dimensional structural reported for only one family member, human hORF6. Sequence alignment of AhpE with the human 1-Cys Prx, hORF6, showed that it is homologous with this protein also, but with lower sequence identity (24%), and that it lacks the C-terminal domain that mediates dimerization of hORF6.
The Hill et al., J. Mol. Cell. Cardiol. (2012) 52:559-567 review showed that the sulfenylated cysteine can react with glutathione (GSH) to form protein-mixed disulfides. A symmetrical set of reactions can also occur in which the thiolate of GSH is activated either by radical abstraction or sulfenic acid formation. Indeed, several proteins, such as hemoglobin, peroxiredoxins, the 20S proteasome, branched-chain aminotransferase, β-actin, inhibitor of nuclear factor κB kinase subunit β (IKK-β) and aldose reductase (AR), have been shown to be glutathiolated via a sulfenic acid intermediate. Thus, proteins can be glutathiolated as a result of activation of protein cysteine residues by sulfenylation.
Most thiols (SH-containing compounds) do not react at physiologically significant rates with hydroperoxides or other reactive species. Protein-bound cysteines are not particularly reactive unless they are embedded in a micro-architecture that facilitates cleavage of the hydroperoxy bond by polarization and proton shuttling as in the thiol peroxidases. Evolution has designed these proteins for highly efficient hydroperoxide reduction. Accordingly, they do not only deserve interest as hydroperoxide-detoxifying enzymes, but also as ideal sensors for a hydroperoxide compound (ROOH).
Yet several proteins, such as Prx and thioredoxin, contain a micro-architecture embedded cysteine that is in the thiolate form that readily reacts with H2O2 to form sulfenic acid (Cys-SOH); the sulfenylated residue can then facilitate glutathiolation reactions. For example, the sulfenic acid form of protein tyrosine phosphatase 1B (PTP1B; EC 3.1.3.48) is rapidly converted to a sulfenyl-amide (Cys-S—N—R), which prevents further oxidation to sulfinic and sulfonic acids and promotes glutathiolation and thiol recovery.
Sulfenic acids can also be stabilized non-covalently by interactions with surrounding residues. Glutathione reductase and NADH peroxidase form stabilized sulfenic acids that can favor thiolation reactions. Aldose reductase forms an apparently stable protein-sulfenic acid at Cys298 during ischemia that is glutathiolated enzymatically by GSTP upon reperfusion. Hence, a stabilized sulfenic acid can impart specificity to protein glutathiolation.
The Seo et al., Proc Natl Adad Sci USA 2009 106(38):16163-16168 paper demonstrated that cancer cells are frequently under persistent oxidative stress due to oncogenic stimulation, increased metabolic activity, and mitochondrial malfunction. Elevated reactive oxygen species (ROS) generation in cancer cells serves as an endogenous source of DNA-damaging agents that promote genetic instability. Mounting evidence (see Roos et al., Free Radic. Biol. Med. 2011 51:314-326; Paulsen et al., ACS Chem. Biol., 2010 5(1):47-62 and references therein) also supports a physiological role for ROS as second messengers in intracellular signaling cascades that control cell growth, proliferation, migration, and apoptosis.
In these pathways, stimulation of various cell surface receptors activates the NADPH oxidase complex to generate a burst of hydrogen peroxide (H2O2). H2O2 modulates signal transduction through chemoselective oxidation of cysteine residues in proteins, thereby altering their cellular function. In cancer cells, increased ROS can generate inappropriate proliferation signals and thus, contribute to tumor growth and other biological events that promote malignancy.
In a microarray screening study, Seo et al., Proc Natl Adad Sci USA 2009 106(38):16163-16168 reported that among the nine patient tumor tissue samples that examined, sulfenic acid abundance varied by up to seven-fold. In addition, six of seven individuals with transitional or squamous cell carcinoma exhibited a significant increase in the extent of sulfenic acid modifications, relative to matched normal tissue (P<0.005 by paired t test); adeno- and epidermoid bladder carcinomas samples were also associated with elevated levels of sulfenic acid (P<0.001 by paired t test). Although the number of paired samples on the array chip is too small to draw broad conclusions, these initial observations suggested to Seo et al. that elevated levels of sulfenic acid might be hallmark of bladder tumor tissues. Consistent with this hypothesis, lower total thiol groups have been reported in the blood plasma of patients with bladder cancer, as compared to healthy controls.
As indicated from the above-mentioned three categories of proteins, some of the proteins examined in the above papers are recognized for their enzymatic activity and have been given “EC” classification under the Enzyme commission report of 1961 and/or the Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, whereas others have not been so designated in the literature. Table A, below, provides names of exemplary proteins and their enzyme classification.
TABLE ABiologicalEnzymeProtein NameActivityClassificationPeroxiredoxinPeroxidaseEC 1.11.1.15(Prxl); AhpEProtein tyrosinePhosphatase;EC 3.1.3.48phosphatase 1BCellularsignalingAldose reductaseReductaseEC 1.1.1.21Orp1Peroxidase;—transcriptionregulatorGAPDHGlucoseEC 1.2.1.12metabolism andtranscriptionregulatorActinCytoskeleton—component; CellsignalingEGFRPhosphorylase;—CellularsignalingNitrile hydrataseNitrile4.2.1.84hydration
Borinic acids, boronic acids, and their derivatives such as their esters, illustrated below with R's=hydrocarbyl, aryl, etc., are known to form dative bonds with biologically relevant alcohols such as ribose (see Baker et al., Science, 2007 316:1759-1761; Baker et. al., J. Med. Chem., 2006 49:4447-4450; Zhu et al., J. Am. Chem. Soc., 2006 128:1222-1232; Nishiyabu et al., Chem. Commun., 2012 47:1106-1123; Ellis et al., J. Am. Chem. Soc., 2012 134:3631-3634; and citations therein) and active site protein serines (see Adams et al., Cancer Invest., 2004 22:304-311; Groll et al., Structure, 2006 14:451-456; Baldwin et al., Bioorg. Med. Chem. Lett., 1991 1(1):9-12). Therefore, as inhibitors, boronic and borinic acids represent a class of compounds with attractive pharmaceutical applications (for a few examples see, Hall, in Boronic acids; Wiley-VCH: Weinheim, 2005 and citations therein; Groziak, Am. J. Therapeutics, 2001 8:321-328; Baker et al., Chem. Soc. Rev., 2011 40:4279-4285; Priestley et al., Bioorg. Med. Chem. Lett., 2002 12:3199-3202; Albers et al., J. Med. Chem., 2011, 54:4619-4626; Baggio et al., J. Am. Chem. Soc., 1997 119:8107-8108; Asano et al., ChemBioChem, 2004 5: 483-490; Baker et al., Science 2007 316:1759-1761; and Baker et. Al., J. Med. Chem., 2006 49:4447-4450).

An underlying concept of this invention is that a contemplated boron-containing compound can form a dative bond (complex) specifically with the sulfenic acid form of a protein. The thiol form of the protein does not interact with the boron-containing compound. This complex formation permits specific trapping of the oxidized form of the protein. For example, in the case of PTP1B, this complex formation results in the trapping of the inactive form of the enzyme, thereby providing a novel mechanism of enzyme inactivation.

Nitrile hydratase provides an even more direct inhibition assay than does PTP. The catalytic activity of nitrile hydratase requires the presence of a catalytic cysteine-sulfenic acid (Cys-SOH). [Hashimoto et al., J. Biol. Chem. 2008 283:36617-36623.] Thus, that enzyme provides a platform for a direct inhibition assay of catalytic activity. As shown hereinafter, the catalytic activity of nitrile hydratase is inhibited in the presence of boronic acids, thereby illustrating the utility of one aspect of the present invention.
Initial reviews of the scientific literature have failed to find any reports of borinic acid and/or boronic acid or their esters, salts, hydrates, or solvates being used for the targeting of protein sulfenic acids. Currently, it is believed that there are no reported therapeutics that specifically targeting the sulfenic acid protein modification.